JATROPHA CURCAS HYBRID NANDAN-2 FOR HIGH OIL CONTENT AND CONSTRUCTION OF MOLECULAR MARKERS SPECIFIC TO IT

Field of invention:

The present invention relates to a new and distinctive Jatropha curcas hybrid, Nandan-2 which is responsible high oil content and construction of molecular marker specific to hybrid Jatropha curcas. In particular it relates to process for construction of a hybrid specific molecular marker responsible for high seed yield and its progeny by all means i.e., Vegetative/ Sexual/ Asexual/ Tissue culture/ Clonal propagation.

Background and prior art:

The present investigation relates to a new and distinctive Jatropha curcas hybrid designated as Nandan-2. All publications cited in this application are herein incorporated by reference.

In India, the tree species considered for biodiesel production are those that are not used as edible oil, which do not complete with food crops for resources and not used in traditional medicine.

Among the oil borne tree species, Jatropha curcas is considered to be the most prospective plant due to its hardiness, rapid growth, easy propagation, drought endurance, high oil content, low gestation period and ability to grow on degraded soils and waste lands with low to high rainfall. Analysis of Jatropha curcas seeds shows the following chemical composition; moisture 6.62; protein 18.2; fat 38.0; carbohydrates 17.30; fiber 15.50; and ash 4.5%. The oil content is 30 to 35% in the seeds and 50 to 60% in the kernel. There is some chemicals element in the seeds which possess poisonous and purgative properties and render the oil non edible for human consumption. The oil is obtained from decorticated seeds by expression or solvent extraction.

Breeding objectives depend on use of the specific crop; increasing yield is a primary objective in all programs. Oil yield of Jatropha curcas is determined by number of inflorescences per plant, number of fertile pistillate flowers per inflorescence, seeds per capsule, 100-seed weight and seed oil content. As the maximum number of seeds per capsule is limited and the agronomic factor of planting density does not offer much flexibility for increasing oil yields, selection should focus on the other yield components and oil content of seed to obtain higher yield. Heritable variation exists for all of these components except number of seeds per capsule, and breeders may directly or indirectly select for increases in any of them.

There are highly significant correlations in different seed samples between the 100-seed weight and percent of crude fat content. This is interesting from the breeders' point of view, as simple selection for high 100-seed weight could imply increased crude fat contents. Therefore, to get more oil yield per hectare development of a high yielding Jatropha curcas hybrid with a high 100-seed weight, and consequently a higher crude fat content is very necessary.

Numerous steps are involved in the development of any novel, desirable cultivar. Plant breeding begins with the analysis and definition of problems and weakness of the current cultivars, followed by the fixation of program goals, and the definition of specific breeding objectives. The next step is selection of parental lines that posse the traits, required to meet the program goals. The goal is to combine in a single cultivar an improved combination of desirable traits from the parental sources. These important traits may include higher yield, resistance to disease and insect pests, better canopy structure, tolerance to environmental stress, better agronomic characteristics and higher oil content in oil bearing plants.

US7,005,298 relates to in vitro micropropagation and phytofortifϊcation of a phytopharmaceutical plant comprising: a) culturing a sterile explant of said phytopharmaceutical plant on an induction medium comprising at least one plant growth regulator having cytokinin activity, to form regenerated tissue; b) transferring said regenerated tissue to a first basal medium lacking said plant growth regulator having cytokinin activity, and culturing to form plantlets; and c) subculturing said plantlets onto a second basal medium supplemented with one, or more than one additive of interest at an amount from about 50 to about 200 mg/L.

US 20070218175 discloses and claims a method of producing a triglyceride solution, comprising dissolving triglyceride in a liquid fatty acid alkyl ester to form a triglyceride solution from and from an oil-bearing plant. US20030028927 discloses method of detecting genetic variation in a plant genome, comprising inhibiting Hsp90 activity in at least one plant cell and method of identifying a plant polymorphism.

US20020194646 discloses a method of creating a transfected or transgenic plant chosen from the group consisting of ornamental, horticultural, forestry, medicinal or Nicotiana sp. plants, exhibiting a dwarf phenotype comprising: expressing in the plant the DNA identified by a polynucleotide sequence chosen from the group consisting of SEQ. ID NO: 1-122 or the mRNA encoded by the DNA identified by a polynucleotide sequence chosen from the group consisting of SEQ. ID NO: 1-122.

WO2006043281 discloses an improved process for the preparation of biodiesel from triglyceride oils through transesterification. More specifically, the invention relates to preparation of fatty acid methyl ester of oil mechanically expelled from whole seeds of Jatropha curcas. The utilization of the co products for preparing value added products and the integrated approach to utilize and manage the effluent streams make the process environmentally friendly.

CNl 799340 discloses a method of mass production grease tung seed sprout through tissue culture which provides a high effective way for the grease tungartificial breeding, germ- free sprout and species of high quality breeding.

Thus the present invention relates to new and distinctive Jatropha curcas hybrid which is responsible for high seed yield and construction of molecular marker specific to hybrid Jatropha curcas.

The goal of Jatropha curcas hybridization is to develop new, unique and superior Jatropha curcas hybrids. The breeder initially selects and crosses two or more parental lines, followed by selection among the many new genetic combinations. The breeder can theoretically generate billions of new and different genetic combinations via crossing. The breeder has no direct control at the cellular level; therefore, two breeders will never develop the same line, or even very similar line, having the same traits of Jatropha curcas. Choice of breeding procedures to select for the improved combination of traits depends on the mode of plant reproduction, the heritability of the traits being improved, and the type of cultivar used commercially. For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants.

As Jatropha curcas is a cross-pollinated crop, any genetic improvement has to be based on populations. Mass selection would be the simplest breeding procedure, where superior selected plants are composited. Populations can be stepwise improved if they remain large, so that additive genetic variation can be used. The process of recurrent selection is widely used in tree breeding. This involves concurrent cycles of selection with or without progeny tests. There are possibilities for the breeder to modify the process. In addition, hybrid cultivars could be bred to use the heterosis effect. The existence of male sterile cultivars would facilitate crossings. Emasculation was not necessary for hybridization in the insect- free greenhouse due to absence of insect vectors and the time lag of anthesis of staminate flowers. The standard routine of bagging should be sufficient in the field. However, to avoid self-pollination if staminate and pistillate flowers were to open simultaneously emasculation could be required as Jatropha curcas is self-compatible and this can be achieved very easily as staminate and pistillate flowers look very distinct.

Jatropha curcas is an important and valuable oil seed crop. Thus, a continuing goal of Jatropha curcas breeders is to develop stable, high yielding hybrid that are agronomically sound. The reasons for this goal are to maximize the amount of seed oil produced on the land used and to supply biofuel. To accomplish this goal, the Jatropha curcas breeder must select and develop Jatropha curcas plants that have the traits that result in superior hybrid.

The two important features of cross-pollinated species are inbreeding depression and heterosis. Population improvement schemes generally aim at keeping inbreeding at a low level to avoid its ill effects, but an effort to exploit heterosis is rarely made. Heterosis is the basis of hybrid varieties and hybrid varieties are the best means for utilizing heterosis. Object of the invention:

The main object of the present invention is to develop new, unique and superior Jatropha curcas hybrids, Nandan-2 and method for construction of a hybrid specific molecular marker responsible for high oil content and its progeny.

Another object of the invention is to provide a process for producing a Jatropha curcas plant by crossing (JCZ-T62 x JCZ-O41) followed by biparental crossing adopting North Carolina Design II which includes hybrid production, crosses to population, clonal propagation, micropropogation and provides regenerable cells for use in tissue culture.

Summary of the invention:

The present invention relates to a hybrid Jatropha curcas plant designated as Nandan-2 responsible for high oil content and construction of hybrid specific molecular markers. The invention is also directed to processes for producing a Jatropha curcas plant by crossing (JCZ-T62 x JCZ-041) followed by biparental crossing adopting North Carolina Design II. Thus, any process using the hybrid Jatropha curcas plant Nandan-2 in backcross, hybrid production, crosses to population, clonal propagation, micropropagation and the like are part of this invention. All plants which are a progeny of or descend from Nandan-2 are within the scope of this invention. It is an aspect of this invention for Jatropha curcas hybrid Nandan-2 to be used in crosses with other, different, Jatropha curcas plants to produce first generation (Fi) Jatropha curcas hybrid seeds and plants with superior characteristics.

In another aspect, the present invention provides regenerable cells for use in tissue culture procedure of Nandan-2. The tissue culture is preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing Jatropha curcas plant, and of regenerating plants having substantially the same genotype as the foregoing Jatropha curcas plant. Preferably, the regeneration cells in such tissue cultures are embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, pistils, root tips, seeds or stems. Still further, the present invention provides Jatropha curcas, Nandan- 2 plants regenerated from the tissue cultures of the invention.

Essentially all the physiological and morphological characteristics means a plant having physiological and morphological characteristics mean a plant having the physiological and morphological characteristics of the cultivar, except for the characteristics derived from the hybridization

• 100-Seed weight: The weight of 100 Jatropha curcas seeds as measured in grams.

• Regeneration: Regeneration refers to the development of a plant from tissue culture.

The following embodiments and aspects thereof are described in conjunction with procedures, tools and processes which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

Description of the Drawings:

Figure 1 Gel electrophoresis of RAPD and PCR analysis for detecting Polymorphism

Figure 2 DNA Sequence of Nandan-2(935 bp)

Detailed Description of the Invention

The Present invention discloses Jatropha curcas hybrid Nandan-2 plants have higher oil content with higher 100-seed weight.

Germplasm accessions of Jatropha curcas have been collected from different agro climatic conditions of the country covering the parts of South, North, West, East & Central India. All these accessions have been collected and put to progeny and provenance trials in known agro-climatic region like Zaheerabad which is situated at 17.8 SN Latitude and 77.39 E Longitude. It has an altitude of 610-660 mts above MSL, with an average rainfall of 926 mm occurring between June and October. The temperature ranges between a maximum of 42°c and a minimum of 26°c. The soil type is laterite, rich in iron. The performances of these germplasm accessions in respect to oil content in seeds and higher 100-seed weight and other morphological characters have been studied for two consecutive years. From these accessions two germplasm were selected for the hybridization program. The female parent (JCZ-T62) was selected for having the character of higher 100-seed weight with near three seeds per capsule. The pollen parent (JCZ-041) was selected due to its higher seed oil content. Stability of the characters identified for selection of both the female and male parents has been established for two consecutive years.

The selected plant were then crossed (JCZ-T62 x JCZ-O41) for the production of Nandan-2. Jatropha curcas hybrid Nandan-2 has the following morphological and other characteristics as mentioned in Table 1, recorded at the age of three years.

This invention also is directed to procedures for producing a Jatropha curcas hybrid by crossing a first parent Jatropha curcas plant with a second parent Jatropha curcas plant, wherein the first or second Jatropha curcas plant is a Jatropha curcas from the hybrid Nandan-2. Further, both first and second parent Jatropha curcas plant may be from the hybrid Nandan-2. Therefore, any procedures using the hybrid Nandan-2 are part of this invention: selfing, backcross, hybrid breeding and crosses to populations. Any plants produced using Jatropha curcas hybrid Nandan-2 as a parent are within the scope of this invention.

This invention also is directed to procedures for producing Jatropha curcas hybrid Nandan-2 with a second Jatropha curcas plant and growing the progeny seed, and repeating the crossing and growing steps with the Jatropha curcas hybrid Nandan-2 derived plant from 0 to 7 times. Thus, any such procedures using the Jatropha curcas hybrid Nandan-2 are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using Jatropha curcas hybrid Nandan-2 as a parent are within the scope of this invention, including plants derived from Jatropha curcas hybrid Nandan-2.

It should be understood that the parents of hybrid Nandan-2 can, through routine manipulation of cytoplasmic or other factors, be produced in a male-sterile form. Such embodiments are also contemplated within the scope of the present claims.

Identification of the Marker Molecular Characterization of Nandan-2

Plant Material

Attempts were made to develop diagnostic molecular markers for the high oil content hybrid Nandan-2. For this purpose, molecular analysis of the hybrid Nandan-2 was carried out with 9 more diverse Jatropha curcas lines that exhibited variability in seed characters (large versus small), maturity pattern (extended flowering versus synchronous maturity), leaf size (small versus medium) and yield (high vs low).

DNA Extraction

Total genomic DNA was extracted from younger leaves of the hybrid (Nandan-2) following the standard CTAB process with minor modifications (Doyle and Doyle 1987). Five grams of leaves were ground in liquid nitrogen, then homogenized in 20 ml of extraction buffer (2% CTAB, 20 mM EDTA, 2% PVP, 1.4 M NaCl, 100 mM Tris-HCl pH 8.0 and 1% β-mercaptoethanol) and incubated at 65 ⁰C for Ih. The supernatant was treated with RNase A (100 μg/ml), incubated at 37 ⁰C for 30 min and twice extracted with chloroform: isoamylalcohol (24:1 v/v). The DNA was precipitated with isopropanol and washed twice with 70% ethanol. The rMleted DNA was air dried and resuspended in 500 μl of sterile Millipore water and stored overnight at -20 ⁰C.

RAPD and PCR analysis

A total of 200 decamer primers from Operon kits - OPB to OPK (Operon technologies, Alameda, USA) were used for DNA amplification according to the process of Williams et al. (1990). The PCR amplification reaction (10 μl) consisted of 2.5 ng of DNA, Ix PCR buffer (10 mM Tris pH 9.0, 50 mM KCl, 1.5 mM MgCl₂), 100 μM of each of the four dNTPs, 0.4 μM of RAPD primer and 0.3 U of Taq DNA polymerase (Bangalore Genei, India). PCR amplifications were performed in an Gene Amp 9700 Thermal Cycler (Eppendorf) with an initial denaturation at 94 ⁰C for 3 min followed by 45 cycles at 94 ⁰C for 45 s, 36 ⁰C for 30 s and 72 ⁰C for 2 min with a final extension at 72 ⁰C for 7 min. The PCR products were separated on 1.5% agarose gel in Ix TAE buffer by electrophoresis at 100 V for 3 h and visualized with ethidium bromide staining under gel documentation system. In general, RAPD markers suffer from a lack of reproducibility, but to check the consistency of the electrophoretic patterns and the polymorphism detected, every PCR reaction was repeated twice. All the PCR amplifications included a negative control (no DNA) to avoid erroneous interpretations. The 200 tested primers gave robust amplification profiles. Polymorphic bands were checked for accession specific bands. Only one marker was found specific to Nandan-2.

Further, the distinction of the hybrid Nandan-2 has been accomplished through development of a molecular marker specific to the hybrid. The molecular marker gives a specific band of 935 bp (M 13 primer) in Nandan-2. The inheritance of the marker was validated by checking it on progeny (20) resulting from this promising hybrid. Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce Jatropha curcas plants having the physiological and morphological characteristics of Jatropha curcas hybrid Nandan-2.

As used herein, the term 'tissue culture' indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant. Exemplary types of tissue cultures are protoplasts, calli, plant meristems, and plant cells that can generate tissue culture that are intact in plants or parts of plants, such as embryos, pollen flowers seeds, inflorescences, leaves, stems, roots, root tips, anthers, and the like. - Means for preparing and maintaining plant tissue culture are well known in the art. By way of example, a tissue culture comprising organs has been used to produce regenerated plants.

As used herein, the term 'plant' includes plant cells, plant protoplasts, plant cells of tissue culture from which Jatropha curcas plants can be regenerated, plant calli, plant meristems, and plant cells that are intact in plants or parts of plants, such as pollen, flowers, embryos, ovules, seeds, inflorescence, leaves, stems, pistils, anthers and the like. Thus another aspect of this invention is to provide for cells which upon growth and differentiation produce a cultivar having essentially all of the physiological and morphological characteristics of Nandan-2.

The present invention contemplates a Jatropha curcas plant regenerated from tissue culture of the hybrid Jatropha curcas plant of the present invention. As is well known in the art, tissue culture of Jatropha curcas can be used for the in vitro regeneration of a Jatropha curcas plant. Tissue culture of various tissues of Jatropha curcas and regeneration of plants there from is well known and widely published. For example, reference may be had to Lin et. al., Plant Physiol Commun 38: 252 (2002); Lu et. al., Environ. Biol. 9: 127 (2003); Sujatha et. al., Plant Cell Tiss. Org. Cult. 44: 135 (1996); and Wei Qin, J. Plant Physiol. MoI. Bio. 30: 475 (2004). Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce Jatropha curcas plants having the physiological and morphological characteristics of Nandan-2.

Another process involves producing a population of Nandan-2 progeny Jatropha curcas plants, comprising crossing Nandan-2 with another Jatropha curcas plant, thereby producing a population of Jatropha curcas plants, which on average, derive 50% of their alleles from Nandan-2. A plant of this population may be selected and repeatedly selfed or sibbed with a Jatropha curcas plant resulting from these successive filial generations. One embodiments of this invention is the Jatropha curcas cultivar produced by this process and that has obtained at least 50 % of its alleles from hybrid Nandan-2. The same process may be used with one or both of the parents of Nandan-2.

Progeny of Jatropha curcas hybrid Nandan-2 may also be characterized through their filial relationship with Nandan-2, as for example, being within a certain number of breeding crosses of Nandan-2. A breeding cross is a cross made to introduce new genetics into the progeny, and is distinguished from a cross, such as a self or a sib cross, made to select among existing genetic alleles. The lower the number of breeding crosses in the pedigree, the closer the relationship between Nandan-2 and its progeny. For example, progeny produced by the procedures described herein may be within 1, 2, 3, 4, or 5 breeding crosses of Nandan-2.

Tables:

The following tables' present data on the traits and characteristics of Jatropha curcas hybrid Nandan-2 as compare to its parental lines JCZ-T62 and JCZ-041.

The results in Table 2 compare the oil yield, number of inflorescences per plant, number of pistillate flowers per plant, female to male flower ratio, seeds per capsule, 100-seed weight, seed oil content of Nandan-2, the hybrid of the recent invention produced by crossing JCZ-T62 and JCZ-041. As shown in Table-2, hybrid Nandan-2 has significant higher oil content, oil yield, more seeds per capsule, and higher 100-seed weight than both of the parents of Nandan-2. In Table 2 below, column 2 shows the oil yield, column 3 shows number of inflorescences per plant, column 4 shows number of pistillate flowers per plant, column 5 shows female to male flower ratio, column 6 shows seeds per capsule, column 7 shows 100-seed weight and column 7 shows seed oil content.

TABLE 1: HYBRID DESCRIPTION INFORMATION

Inflorescence type Compact

Female to male ratio 1 : 11

Flower color Light green

Capsule size (diameter) Medium (2 8 - 3.2 cm)

Pedicel length 1.53 cm

Seed characters

Seed coat color Black

Shape class (Length / width ratio) Long

Size (measurements) Length ' Width L / W

17.5 mm 11.4 mm 1.54

100-seed weight 62 g

Oil content 38 %

Disease resistance

Powdery mildew (Erysiphe euphorbiae) Moderately resistant

Insect pest resistance

Inflorescence & capsule borer (Pempelia Moderately resistant morosalis) Moderately resistant

Leaf miner (Neurobathra curcassi Busck.) Moderately susceptible

Bugs {Scutellera nobilis Fabr.)

TABLE 2